Extraction of materials from regions of interest in a sample

ABSTRACT

A system for extracting material from a region of interest includes a fluid delivery base comprising an inlet channel and an outlet channel formed within the fluid delivery base; a gasket affixed to the fluid delivery base, wherein the gasket comprises at least one opening exposing an open end of the inlet channel and an open end of the outlet channel; a support comprising a sample-supporting surface facing the gasket and an opposing surface; and an alignment member coupled to the opposing surface in a fixed position and such that the support is positioned between the fluid delivery base and the alignment member, wherein one or both of the alignment member or the fluid delivery base are biased towards one another by a force (e.g., a magnet or spring force) and wherein the fluid delivery base is separable from the support and configured to move along a plane of the sample-supporting surface to align with the alignment member.

BACKGROUND

The subject matter disclosed herein relates to techniques for extractingbiological molecules from an existing sample, such as a pathology slide.

Medical researchers often obtain patient samples, such as biopsies, andpreserve such samples as pathology slides, core samples, etc., fordiagnosis and visualization. When such samples contain particularregions of interest, the researchers may wish to extract materials fromthese regions of interest for additional studies. For example,researchers may examine a slide including both tumor and normal cells,and may wish to extract DNA from only the tumor cells in the slide toassess the DNA for the presence of particular mutations. However,extraction of material from only a region of interest and withoutdamaging the material is complex. For example, in laser capturemicro-dissection, a focused laser beam ablates tissue to define a regionof interest but damage the surrounding material. Other techniques mayinvolve tissue encapsulation, which introduces an additional material tothe sample.

BRIEF DESCRIPTION

Certain embodiments commensurate in scope with the originally claimedinvention are summarized below. These embodiments are not intended tolimit the scope of the claimed invention, but rather these embodimentsare intended only to provide a brief summary of possible forms of theinvention. Indeed, the invention may encompass a variety of forms thatmay be similar to or different from the embodiments set forth below.

In one embodiment, a system for extracting material from a region ofinterest includes a fluid delivery base. The system also includes afirst channel within the fluid delivery base and terminating at a firstchannel end at a sample-facing surface of the fluid delivery base,wherein the first channel comprises a first channel opening configuredto couple to a fluid inlet to fluidically couple the fluid inlet thefirst channel end and a second channel within the fluid delivery baseand terminating at a second channel end at the sample-facing surface ofthe fluid delivery base, wherein the second channel comprises a secondchannel opening configured to couple to fluid outlet to fluidicallycouple the fluid outlet to the second channel end. The system alsoincludes a gasket coupled to the sample-facing surface and comprising agasket opening aligned with an area of the sample-facing surfacecomprising the first channel end and the second channel end. The systemalso includes a support comprising a sample-supporting surface facingthe gasket and an opposing surface and an alignment member coupled tothe opposing surface, wherein the fluid delivery base is separable fromthe support and configured to move along a plane of thesample-supporting surface to align with the alignment member.

In another embodiment, a method of extracting material from a region ofinterest includes the steps of aligning one or more fluid channels of afluid delivery base with an alignment member to define a region ofinterest on a sample, wherein the fluid delivery base and the alignmentmember are positioned on opposing surfaces of the sample such that thefluid delivery base and the alignment member are separated from oneanother by at least the sample and wherein the aligning compriseschanging a position of the fluid delivery base or the alignment memberrelative to one another; delivering extraction fluid to the sample viathe one or more fluid channels; and collecting the extraction fluid viathe one or more fluid channels.

In yet another embodiment, a system for extracting material from aregion of interest includes a fluid delivery base comprising one or morechannels formed within the fluid delivery base, wherein the fluiddelivery base comprises a metal; a gasket affixed to the fluid deliverybase, wherein the gasket comprises at least one opening exposing an openend of at least one of the one or more channels; a support comprising asample-supporting surface facing the gasket and an opposing surface; andan alignment member coupled to the opposing surface in a fixed positionand such that the support is positioned between the fluid delivery baseand the alignment member, wherein the fluid delivery base is separablefrom the support and configured to move along a plane of thesample-supporting surface to align with the alignment member.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 is a partial cross-sectional view of an extraction system inaccordance with embodiments of the present techniques;

FIG. 2 is a partial cross-sectional view of the extraction system ofFIG. 1 showing the isolation area on the sample;

FIG. 3 is a top view of an exemplary extraction system showing multipleregions of interest on the sample;

FIG. 4 is a schematic view of a magnetic field of a magnetic fluiddelivery base and a magnetic alignment member in accordance withembodiments of the present techniques;

FIG. 5 is a cross-sectional view of a fluid delivery base in accordancewith embodiments of the present techniques;

FIG. 6 is a perspective view of an extraction system in contact with asample in accordance with embodiments of the present techniques;

FIG. 7 is a perspective view of an alternative extraction system incontact with a sample in accordance with embodiments of the presenttechniques;

FIG. 8 is a partial cross-sectional view of an extraction systemincluding an internal light in accordance with embodiments of thepresent techniques;

FIG. 9 is a schematic representation of a technique for aligning theseparable fluid delivery base of an extraction system with a fixedalignment member in accordance with embodiments of the presenttechniques;

FIG. 10 is a schematic representation of a technique for aligning theseparable fluid delivery base of an extraction system with a removablealignment member in accordance with embodiments of the presenttechniques;

FIG. 11 is a side view of an extraction system including an adjustablearm in accordance with embodiments of the present techniques;

FIG. 12 a partial cross-sectional view of a multi-region of interestextraction system in accordance with embodiments of the presenttechniques;

FIG. 13 is a schematic representation of a technique for aligning thefluid delivery base of a fluid extraction device with a sample on anx-y-z stage in accordance with embodiments of the present techniques;

FIG. 14 is a stained image of a colon adenocarcinoma with the top panelshowing DNA staining and including background, the middle panel showingstaining with the background removed and the bottom panel after regionof extraction removal; and

FIG. 15 is stained image of a breast invasive ductal carcinoma with thetop panel showing DNA staining and including background, the middlepanel showing staining with the background removed and the bottom panelafter region of extraction removal.

DETAILED DESCRIPTION

Researchers may wish to extract regions of interest from biological orenvironmental samples with minimal disruption of the original slide orsection. A region of interest may be any user-defined region on asample, and may be a single cell, a subcellular region, or amulticellular region of a sample. Regardless of the region of interestfor the user, techniques used for extracting regions of interest fromthe sample may damage the surrounding sample and/or may be expensive orcomplex. For example, laser-based techniques may be used that cut arounda particular region of interest, but such techniques may involveexpensive equipment, skilled technicians, and long processing times. Forexample, laser capture microdissection may involve complex associatedsample preparation and is performed by skilled technicians Othertechniques target a region of interest via transfer, such as applyingliquid wax on a slide and removing the wax after solidification.However, such techniques are difficult to automate and provide onlylimited spatial resolution, in certain cases because the wax isdifficult to limit to a particular location with high resolution. Inanother example, extraction may be achieved by physically scraping (andwasting) away all of the non-ROI tissue. Such physical extractions areassociated with a destruction of the remaining sample, which preventsfurther analysis or sample mapping.

Provided herein is a self-aligning region of interest extractiontechnique that provides improved spatial resolution without complexequipment. In addition, the present techniques provide high extractionefficiencies for materials of interest within the sample, such asnucleic acids, without damaging the surrounding tissue. In turn,surrounding sample preservation allows for more complex analysis to beperformed on a sample, such as mapping or heterogeneity analysis. Thepresent techniques are also suitable for automation or higherthroughput.

The disclosed techniques may be used in conjunction with samples ofbiological materials. As used herein, the term “biological material”refers to material obtained from samples of a biological subject,including biological tissue or fluid obtained from a subject. Suchsamples may be, but are not limited to, body fluid (e.g., blood, bloodplasma, serum, or urine), tissues, fractions, and cells isolated from,or located in, any biological system, such as mammals. Biologicalsamples and/or biological materials also may include sections of thebiological sample including tissues (e.g., sectional portions of anorgan or tissue). Biological samples may also include extracts from abiological sample, for example, a population of cells from a biologicalfluid (e.g., blood or urine). In certain embodiments, the biologicalmaterial may include proteins, nucleic acids, carbohydrates, fattyacids, and/or small molecules. It should be understood that the samplesmay be histological samples, pathology samples, or tissue core samplesand may be in the form of slides, sections, multi-well plates, etc.Further, the disclosed techniques may also be used in conjunction withnon-biological samples, environmental samples, or forensic samples.

Turning to FIG. 1, the extraction system 10 as provided herein includesa fluid delivery base 12 that, in operation, is positioned to deliverextraction fluid directly to the sample 14. The sample 12 is positionedon a top surface 16 of a support 20. A bottom surface 22 of the support20 opposes the top surface and is coupled to or includes an alignmentmember 24. In operation, the fluid delivery base 12 is applied to thesample 14 by operator or machine manipulation. In one embodiment, thealignment member 24 and the fluid delivery base 12 self-align undermagnetic force to hold the fluid delivery base 12 in position on thesample 14. In certain embodiments, one or both of the fluid deliverybase 12 or the alignment member 24 is a magnet, e.g., a permanent magnetor an electromagnet. Such that the fluid delivery base 12 and thealignment member 24 magnetically align to hold the fluid delivery base12 in place.

When the fluid delivery base 12 is aligned or correctly positioned onthe sample 14, a gasket 30 on a sample-facing surface 32 of the fluiddelivery base 12 comes into direct contact with the sample. Once aportion of the sample is isolated via the gasket 30, fluid delivery forextraction may take place. As depicted, the fluid delivery base 12 mayinclude a fluid inlet channel 34 for delivering an extraction fluid,represented by arrow 36, to the isolated sample portion. Further, thefluid delivery base may include a fluid outlet channel 38 for collectingthe incubated extraction liquid and recovered materials e.g., biologicalmaterials, represented by arrow 40.

The fluid delivery base 12 may include the channels 34 and 38 asintegrally formed passageways within the body of the fluid delivery base12. For example, the channels 34 and 38 may be formed via drilling or aspart of an injection mold die. While the depicted embodiment includes asingle inlet channel 34 and outlet channel 38, it should be understoodthat there may be any suitable number of channels 34 and 38, and thatthe channels 34 and 38 may be present in equal or unequal numbers.Further, the size (e.g., inner diameter) of each channel 34 or 38 may beselected based on a desired region of interest size. That is, forrelatively small regions of interest, the channels 34 and 38 may beformed with correspondingly small inner diameters. In certainembodiments, the inlet and outlet functions may be achieved via a singlechannel used for inflow and outflow. Further, while the channels 34 and38 may be formed within the fluid delivery base, in other embodiments,the fluid delivery base 12 may form a central passage (e.g. may bedonut-shaped) that facilitates insertion of preformed channels 34 and 38within the passageway. Such an embodiment may help keep the fluiddelivery base 12 isolated from contact with biological materials, whichmay in turn facilitate reuse.

The inlet channel 34 terminates at channel end 44 and the outlet channel38 terminates at channel end 46 on the sample-facing surface 32 andwithin an area 48 defined by the gasket 30. In the depicted embodiment,the channels 34 and 38 are substantially parallel to one another andopen at respective channel ends 50 and 52 disposed on a top surface 54of the fluid delivery base 12. However, in other embodiments, thechannels 34 and 38 may open on a side surface 58 and/or open at aspecialized junction to accommodate couplings to upstream or downstreamtubing. Further, the inlet and outlets channels 34 and 38 may be angledor nonparallel depending on the desired configuration of the fluiddelivery base 12.

The fluid delivery base 12, in certain embodiments, is held in place onthe sample 14 via magnetic force. FIG. 2 is a partial cross-sectionalview of the system 10 showing a region of interest that may be isolatedby the fluid delivery base 12. In operation, the gasket 30 makes directcontact with the sample material 60 at a contact area 62 to isolate aregion of interest 64. When the gasket 30 makes direct contact, theextraction fluid from the inlet channel 34 is sealed from lateraldiffusion across the sample material 60, which facilitates targetedextraction and recovery of the extracted material via outlet channel 38.Further, because the force on the sample 14 is a combination of thegasket sealing force and the magnetic pull force, the applied force maybe selected to minimize pressure of the gasket on the sample material60. To that end, the gasket may be formed from a relatively compliantsealing material, such as a compressible polymer. In particularembodiments, the gasket 30 may be hydrophobic to discourage any lateraldiffusion of extraction fluid.

The fluid delivery base 12 and, in particular, the channels 34 and 38may be configured based on a desired spatial resolution of the region ofinterest 64. In certain embodiments, the region of interest 64 may beless than or larger than 1 mm² or may be on the order of 100 microns orless. In other embodiments, the region of interest 64 may severalcentimeters square. Further, the region of interest may have any desiredshape, including a circle, square, etc. The gasket 30 may be cut to anopening that defines the desired region of interest size and shape. Inone embodiment, the gasket 30 may be cut to an opening that is definedby the system 10. For example, the system 10 may include imagingsoftware under processor control. The user may view the sample 14 via auser interface and may define the region of interest 64 by providinginputs on the user interface, which may be viewed as a superimposedregion on the sample image. Further, the gasket 30 may be selected basedon the region of interest 64 or may be custom cut.

FIG. 3 is an example of a sample 14 showing region of interests that maybe isolated by the fluid delivery base as provided herein. In certainembodiments, the system 10 provides for regions of interest 64 thatencompass an edge or border of the sample material 60. For example, aclinician may wish to determine if cells on a border region havedifferent characteristics than cells in the interior of the sample. Thefluid delivery base is capable of isolating and sealing cells within theregion of interest 64 even if the region of interest includes an edge,which provides advantages over other techniques that may not be able toisolate edge material. Further, the disclosed techniques may be usedwith poor quality samples that were either prepared poorly and/or thathave degraded.

FIG. 4 is a schematic diagram of an embodiment in which both the fluiddelivery base 12 and the alignment member 24 are magnets with a magneticfield (e.g., magnetic fields 70 and 72). For example, the fluid deliverybase 12 is a bar magnet with a south pole 80 oriented towards the regionof interest. The alignment member 24 may be oriented so that its northpole 82 is closest to the south pole 80 of the field delivery base 12 sothat the fluid delivery base 12 and the alignment member 24 will attractone another as they are positioned closer together (by movement of oneor both of the fluid delivery base 12 or the alignment member 24) tohold the fluid delivery base 12 on the region of interest 64. As shown,the alignment may occur along an axis 86 through the alignment member24, the region of interest 64, and the fluid delivery base 12.Generally, the magnetic field 70 may extend beyond the region ofinterest 64 and towards the alignment member 24 when the fluid deliverybase 12, via the gasket 30, is in direct contact with the sample 14. Inaddition, in such a configuration, the magnetic field of the alignmentmember 24 may extend beyond the region of interest 64 and towards thefluid delivery base 12.

It is contemplated that, in embodiments of the present techniques, boththe fluid delivery base 12 and the alignment member 24 are magnets.However, in other embodiments, only one of the fluid delivery base 12 orthe alignment member 24 is a magnet while the other is formed of orincludes a ferrous material. Further, the fluid delivery base 12 or thealignment member 24 may be a permanent magnet, including iron, nickel,cobalt, a rare earth metal magnet, lodestone, a magnetic compositeformed from a metallic magnetic material and a ceramic material orresin, a nanomagnet, etc. The fluid delivery base 12 or the alignmentmember 24 may also include an electromagnet.

FIG. 5 illustrates a cross-section of an implementation of a fluiddelivery base 12 that may include a non-metallic or non-magnetic portion90 and a metallic portion 92. In a particular embodiment, the metallicportion 92 may be magnetic. In the depicted embodiment, the metallicportion 92 is positioned towards the sample end of the fluid deliverybase 12, (i.e., in contact with the gasket 30), but in otherembodiments, the metallic portion 92 may form an exterior cylinder orcasing for the fluid delivery base 12 while the core portion, includingthe fluid channels, may be formed of a non-metallic or non-ferrousmaterial.

The configuration of the fluid delivery base 12 and the alignment member24 (see FIG. 2) may be selected to achieve sufficient pull force to holdthe fluid delivery base 12 without applying so much pressure as todamage the underlying sample when in operation. The magnet pull forcemay be influenced by the materials of the fluid delivery base 12 and thealignment member 24 (i.e., the magnet strength of certain materials ishigher than other materials), and the size of the magnetic material. Forexample, the height (represented by d1) and width (represented by d2) aswell as the depth of the fluid delivery base 12 or the alignment member24 may influence the pull force. In one embodiment, the pull force, case1, of the fluid delivery base, is less than 1 lb., e.g., between about0.32 lb-0.8 lb. However, it should be understood that the pull force isrelated to the size of the magnet, and larger magnets may have largerpull forces. In one embodiment, a ring magnet having an inner diameterof about 0.06 inches and an outer diameter of about 0.2 inches and aheight dimension of about 0.2 inches has a pull force, case 1, of about0.32 lbs, a pull force, case 2, of about 0.38 lbs, and a pull force,case 3, of about 0.63 lbs may be used. A ring magnet having an innerdiameter of about 0.125 inches and an outer diameter of about 0.25inches and a height dimension of about 0.25 inches has a pull force,case 1, of about 0.64 lbs, a pull force, case 2, of about 0.80 lbs, anda pull force, case 3, of about 1.13 lbs may be used. In anotherembodiment, the pull force, case 1, is such that an operator may easilymanually manipulate the fluid delivery base 12 relative to the sample 14during removal or application. In the disclosed embodiments, the case 1pull force is the pull force to remove a magnet from a steel plate, thecase 2 pull force is the pull force to remove a first steel plate from amagnet with a second steel plate on an opposing face, and case 3 is thepull force to remove one magnet from another magnet.

In the depicted embodiment, the fluid delivery base 12 may include a mixof materials to form a shape that is sufficiently-sized to be gripped byan operator but is not so magnetically strong as to damage theunderlying sample. That is, by mixing in materials that are relativelylightweight and nonmetallic, the fluid delivery base 12 may be madelarger without being too heavy or without too strong of a magneticfield.

FIG. 6 is an example of an implementation of the system 10, includingthe fluid delivery base 12 in place on a sample 14. The fluid deliverybase is coupled to inflow tubing 100 and outflow tubing 102 via anadaptor 106. The adaptor 106 may include interior channels that coupleto the inlet and outlet channels 34 and 38 within the fluid deliverybase and exterior channels 110 and 112 to couple to the inflow tubing100 and outflow tubing 102. It is contemplated that all or part of theportion of the system 10 above the sample 14 (e.g., in contact with thesample 14) may be disposable. For example, in one embodiment, the gasket30 may be removable from the fluid delivery base 12, which may becleaned and retained for additional uses while the gasket 30 isdiscarded. In addition, the adaptor 106 and the tubing 100 and 102 maybe provided as a disposable attachment to the fluid delivery base.Alternatively, the fluid delivery base 12 may be assembled as a unitaryassembly with the gasket 30 and the adaptor 106 fixed in place on (e.g.bonded or adhered to) the fluid delivery base 12. In another embodiment,the tubing may couple directly to the fluid delivery base 12 without anadaptor 106 or fluid reservoir. Accordingly, in one embodiment, a sampleextraction kit may include a fluid delivery base 12 with a gasket 30 andan adaptor 106 in place on the fluid delivery base 12. The kit may, incertain embodiments, also include sections of tubing that may beattached, whether during assembly or by an end user, to the exteriorchannels 110 and 112 to form inflow tubing 100 and outflow tubing 102.

Further, in particular embodiments, such a kit may also include one ormore materials for performing an extraction from a sample 14. Forexample, if the sample is a paraffin-embedded tissue section on a glassslide, the kit may include proteinase K for nucleic acid extraction. Inone embodiment, an adaptor may be packed with a suitable amount ofproteinase K to apply to the sample 14 via the fluid delivery base 12.The kit may also include a selection of gaskets 30 with differentopening sizes that facilitate extraction of different-sized regions ofinterest. An operator may select a gasket 30 with an openingcorresponding to the desired region of interest size and apply thegasket 30 prior to the extraction. The gaskets 30 may beremovable/replaceable, and may be peeled away and discarded after use.

FIG. 7 is an example of an implementation of the fluid delivery base 12in place on a sample 14 in which a single channel is used for the outletchannels within the fluid delivery base. A coupler including the outlet114 is fluidically coupled to tubing 116. The sample may be loaded intoan integral reservoir in the fluid delivery base 12. In operation, theuser loads the buffer into the reservoir, which is then pulled to thesample 14. In addition, the sample 14 is shown with sample material 60having already been isolated from a region of interest 64 (shown ashaving the sample material removed in a complete circle) and in place ona second region of interest.

In the various embodiments of the system disclosed herein, the isolationand extraction of material from a particular region of interest 64 maybe performed in conjunction with enzyme or chemical delivery to theregion of interest 64, e.g., via inlet channel 34 or the coupler 114 tofacilitate liquefication of the sample material 60 and subsequentextraction. By performing the extraction on a region of limited size,the extraction workflow may be improved. For example, depending on theenzyme delivered to the region of interest and the temperature of thesample 14, the incubation time may be on the order of seconds (e.g.,less than 10 seconds) rather than several minutes as in othertechniques. In particular, more rapid extraction times may be achievedby agitating the fluid including a buffer and enzyme. Increasedtemperature may also reduce extraction times. However, longer incubationtimes are also contemplated. Any suitable extraction enzyme may be used,such as proteinase K. In other embodiments, the fluid delivery base 12may be used for liquefication and extraction that is not chemically orenzymatically mediated.

FIG. 8 illustrates a cross-section of an implementation of a fluiddelivery base 12 that may include a light source. In operation, as thebase 12 moves towards the sample, the emitted light from the lightsource may help identify the region of interest 64 to assist in properplacement of the fluid delivery base 12 and subsequent materialextraction from the sample. For example, the fluid delivery base 12 mayinclude one or more dedicated light channels 120 coupled to a lightsource 122 (e.g., an LED), which may be on or in the fluid delivery base12 or may be external to the base 12. Optical fibers may be disposed inthe one or more light channels 120, or the channel 120 itself may form alight pipe. Such an arrangement may be advantageous because therelatively small footprint of an optical fiber or light pipe may helpthe spatial resolution of the region of interest 64, e.g., for regionsof interest that are less than 1 mm₂. However, in cases where the regionof interest is larger, the light source may be directly disposed on orin the sample-contacting surface 32 (see FIG. 1) fluid delivery base 12.

The fluid delivery base 12 as provided herein may be used in conjunctionwith a fixed or separable alignment member 24. FIG. 9 is a schematicflow diagram of a technique 200 for using the system 10 with thealignment member 24 fixed in place on (e.g., integral with, adhered to,or part of) the support 20. For example, in certain embodiments, it maybe more convenient and provide more uniform results to have thealignment member 24 fixed in place on the support 20 or sample platform.At step 202, the operator positions the sample 14 on the support 20 at asample placement location such that the region of interest 64corresponds with a location of the alignment member 24 on the opposingsurface 22. In such embodiments, the support 20 may include an indicatoror marking that indicates the sample placement location. At step 204,the fluid delivery base 12 is applied to sample 14 to isolate the regionof interest 64 and the alignment member 24 and the fluid delivery base12 self-align (e.g., the alignment is supported by the strength of themagnetic attraction between these components). Once aligned, theextraction liquid may be introduced at step 206 for incubation andcollection of materials extracted from the region of interest.

FIG. 10 is a schematic flow diagram of an alternate technique 210 forusing the system 10 with a movable alignment member 24. At step 212, theoperator positions the sample 14 on the support 20. At step 214, theoperator either positions the alignment member 24 on the opposingsurface 22 or, alternatively, activates an alignment member that isfixed in place. In one embodiment, the system 10 may include multiplealignment members 24 but may only activate (e.g., apply current toactivate an electromagnet) one of the set. Accordingly, the sampleplacement location would correspond to the active alignment member. Suchan embodiment may permit relatively small alignment members 24 to beused to improve spatial resolution. Once aligned at step 216, theextraction liquid may be introduced at step 218 for incubation andcollection of materials extracted from the region of interest.

While the fluid delivery base 12 and/or the alignment member 24 may bepositioned manually, the system 10 may also be used in conjunction witha mechanical manipulator. For example, FIG. 11 is a side view of anextraction device 256 that includes a mechanical arm 250 and coupled toa base holder 252 that holds the fluid delivery base 12. The base holder252 changes position relative to the mechanical arm 250 (either bymoving or by staying in place when the mechanical arm 250 is moved) toposition the fluid delivery base 12 in close proximity to and on thesample 14. Further, while certain embodiments, of the disclosedtechniques relate to magnetic alignment of the fluid delivery base onthe sample, the fluid delivery base 12 may also be held in place withother biasing forces, e.g., spring force.

The extraction device 256 may also be coupled to a controller 260 thatfacilitates image analysis of the sample (e.g., sample 14) andmovement/alignment of the fluid delivery base at a selected region ofinterest. Accordingly, the extraction device 256 may include an imager264 that detects signals and converts the signals to data that may beprocessed by downstream processors. The imager 264 may operate inaccordance with various physical principles for creating the image dataand may include a fluorescent microscope, a bright field microscope, ordevices adapted for suitable imaging modalities. In general, however,the imager 264 creates image data indicative of the sample 14

The imager 264 and/or the extraction device 256 and mechanical arm 250operate under the control of system control circuitry 262. The systemcontrol circuitry 262 may include a wide range of circuits, such asillumination source control circuits, timing circuits, circuits forcoordinating data acquisition in conjunction with sample movements,circuits for controlling the position of light sources and detectors andthe fluid delivery base 12, and so forth. In the present context, thesystem control circuitry 262 may also include computer-readable memoryelements, such as magnetic, electronic, or optical storage media, forstoring programs and routines executed by the system control circuitry262 or by associated components of the system 10. The stored programs orroutines may include programs or routines for performing all or part ofthe present technique.

Image data acquired by the imager 256 may be processed by the imager 12,for a variety of purposes, for example to convert the acquired data orsignal to digital values, and provided to data acquisition circuitry266. The data acquisition circuitry 266 may perform a wide range ofprocessing functions, such as adjustment of digital dynamic ranges,smoothing or sharpening of data, as well as compiling of data streamsand files, where desired. The data acquisition circuitry 266 may alsotransfer acquired image data to data processing circuitry 270 whereadditional processing and analysis may be performed. The controller 260may include one or more processor-based components, such as generalpurpose or application-specific computers. In addition to theprocessor-based components, the computer may include various memoryand/or storage components including magnetic and optical mass storagedevices and/or internal memory, such as RAM chips. The memory and/orstorage components may be used for storing programs and routines forperforming the techniques described herein that are executed by theoperator workstation or by associated components of the system 10.Alternatively, the programs and routines may be stored on a computeraccessible storage medium and/or memory remote from the operatorworkstation but accessible by network and/or communication interfacespresent on the computer. In one embodiment, the controller 260 mayfacilitate operator selection of a region of interest 64 (e.g., via auser interface) on an image of the sample 14 acquired by the image 264and displayed via the user interface. The controller 260 may alsocontrol movement of the fluid delivery base 12 via the mechanical arm250 to position the fluid delivery base 12 on the region of interest 64.The controller 260 may also control fluid inflow and outflow, and mayinclude one or more settings for controlling flow rate and/or incubationtime.

The extraction techniques disclosed herein may also be used in parallel,as shown in FIG. 12, which illustrates a fluid delivery base 12 capableof isolating multiple regions of interest 64 in parallel. Such aconfiguration may be used for sample mapping, and the system 10 mayextract biological materials from multiple locations on a sample slidefor further analysis. Accordingly, in certain embodiments, thecontroller 260 (FIG. 11) may be used to define multiple regions ofinterest. Alternatively, the positioning may occur manually. FIG. 13 isa schematic view of a technique 300 for manipulating the fluid deliverybase 12 relative to the sample 14 using the fluid extraction device 256.The operator views selects or view automatically selected regions ofinterest superimposed on an acquired image on a workstation 302, whichmay be coupled to the controller 260 (see FIG. 11). The operator mayprovide inputs to select or confirm the regions of interest, whichactivates movement of a stage holding the sample 14, the fluid deliverybase 12, or both as indicated by arrow 310 to correctly align the fluiddelivery base 12 on the region/s of interest. The stage may be an x-y-zstage with freedom of movement in the x, y, and z directions. Further,the fluid delivery base 12 may also include an actuator or stage in thex, y, and/or z direction. In the case of multiple regions of interest64, the fluid delivery base may be positioned concurrently on all ofthem (with associated separate isolation gaskets 30), or may be operatedto align with each in series. Fluid extraction, represented by arrow312, occurs once the fluid delivery base 12 is positioned in place. Thealignment may occur via an imaging or registration step in which thecoordinates of the region of interest 64 are acquired during imaging andthe fluid extraction device is controlled to position the fluid deliverybase 12 at the appropriate coordinates.

Selection of the regions of interest 64 may be coupled to the imageacquisition. For example, the sample 14 may be stained with one or morestains specific for biological markers. The regions of interest 64 mayinclude the regions that are positive for the biological markers. Thecontroller 260 may be configured to align the fluid delivery base 12with the areas of the sample 14 positive for biological markers ofinterest. In this manner, biomolecules, cells, and/or regions expressingspecific proteins or markers may be extracted from the sample 14 forfurther analysis. In one embodiment, an operator may select thebiomarker of interest via the workstation 302 and the controller 260 mayautomatically extract regions including the biomarker.

The following is an example of an extraction performed with anextraction system, such as the systems disclosed herein. For region ofinterest extractions on colon tissue, the extraction diameter was 2 mm.Table 1 shows the extraction results:

TABLE 1 Extraction of Colon Tissue Sample 1 Sample 2 Sample 3 Sample 4DNA (ng/uL) 6.70 6.82 7.70 9.13 Total DNA Yield (ng) 36.83 34.08 42.3743.83 DNA (ng)/mm{circumflex over ( )}2) 11.73 10.86 13.49 13.96

The following is an example of an extraction performed with anextraction system, such as the systems disclosed herein. FIG. 14 showsimages before and after extraction. Table 2 shows the extractionresults:

TABLE 2 Extraction of Colon Adenocarcinoma Sample 1 Sample 2 DNA (ng/uL)(w/background) 8.03 10.57 DNA (ng/uL) (w/o background) 7.30 9.75Extraction Volume 5.6 5.2 Total DNA Yield (ng) 40.91 50.74

The following is an example of an extraction performed with anextraction system, such as the systems disclosed herein. FIG. 15 showsimages before and after extraction. Table 3 shows the extractionresults:

TABLE 3 Extraction of Breast Invasive Ductal Carcinoma. Sample 1 Sample2 DNA (ng/uL) (w/background) 9.71 15.76 DNA (ng/uL) (w/o background)8.65 14.27 Extraction Volume 5.4 5.2 Total DNA Yield (ng) 46.71 74.23

Technical effects of the invention include rapid isolation of regions ofinterest in a biological material without introducing a foreign materialto the remainder of the sample and without wasting or damaging theremainder. The fluid delivery base as disclosed is configured to isolatea region of interest for extraction by self-aligning with othercomponents of the system. Further, the extraction system provided hereinprovides isolation of the sample to prevent sample and/or caregivercontamination. By providing a platform for sealing a region of interest,liquefying the isolated contents of the region of interest, andrecovering the liquefied material, the sample recovery is faster andmore efficient relative to other techniques. In addition, the region ofinterest is isolated from other areas of the sample, preserving theremaining sample for further study.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal languages of the claims.

The invention claimed is:
 1. A system for extracting material from aregion of interest, comprising: a fluid delivery base; a first channelwithin the fluid delivery base and open to a first channel end at asample-facing surface of the fluid delivery base, wherein the firstchannel comprises a first channel opening configured to couple to afluid inlet to fluidically couple the fluid inlet to the first channelend; a second channel within the fluid delivery base and terminating ata second channel end at the sample-facing surface of the fluid deliverybase, wherein the second channel comprises a second channel openingconfigured to couple to a fluid outlet to fluidically couple the fluidoutlet to the second channel end; an adaptor coupled to the firstchannel opening and the second channel opening, wherein the adaptorcomprises an interior channel that couples the first channel end to thefluid inlet; a gasket coupled to the sample-facing surface andcomprising a gasket opening aligned with an area of the sample-facingsurface comprising the first channel end and the second channel end; asupport comprising a sample-supporting surface configured to hold asample against the gasket and an opposing surface; and an alignmentmember coupled to the opposing surface, wherein the fluid delivery baseis separable from the support and configured to move along a plane ofthe sample-supporting surface to align with the alignment member.
 2. Thesystem of claim 1, wherein one or both of the alignment member or thefluid delivery base comprises a magnet.
 3. The system of claim 2,wherein one or both of the fluid delivery base or the alignment membercomprises a ferrous material.
 4. The system of claim 1, wherein thefluid delivery base or the alignment member comprises a permanent magnethaving a magnetic field that extends beyond the opposing surface of thesupport when the gasket is in contact with a sample disposed on thesample-supporting surface.
 5. The system of claim 1, wherein one or bothof the alignment member and the fluid delivery base are biased towardsone another with a spring force.
 6. The system of claim 1, wherein thealignment member is affixed to the support.
 7. The system of claim 1,wherein the alignment member is configured to move relative to theopposing surface of the support.
 8. The system of claim 1, wherein thesupport comprises a heating element.
 9. The system of claim 1, whereinone or both of the fluid delivery base or the alignment member comprisesan electromagnet coupled to a source of electric current.
 10. The systemof claim 1, wherein the first channel and the second channel aresubstantially parallel to each other.
 11. The system of claim 1, whereinthe first channel opening and the second channel opening are disposed ona top surface of the fluid delivery base opposing the sample-facingsurface.
 12. The system of claim 1, wherein the fluid delivery basecomprises a light source configured to emit light from the sample facingsurface within the gasket opening.
 13. The system of claim 12, whereinthe light source is a fiber optic light channel disposed within a thirdchannel in the fluid delivery base.
 14. The system of claim 13, whereinthe third channel is between the first channel and the second channel.15. The system of claim 1, wherein the fluid delivery base and thealignment member, when in operation, align along an axis.
 16. The systemof claim 15, wherein the axis intersects the gasket opening.
 17. Thesystem of claim 1, comprising the fluid inlet and the fluid outlet. 18.The system of claim 1, wherein the gasket opening defines an area lessthan 1 mm².
 19. The system of claim 1, wherein the gasket openingdefines an area greater than 1 mm².
 20. The system of claim 1, whereinthe gasket is positioned between the fluid delivery base and thesample-contacting surface.
 21. The system of claim 1, wherein, inoperation, the gasket contacts the sample.
 22. The system of claim 1,wherein the opposing surface does not contact the sample.
 23. The systemof claim 1, wherein the sample-contacting surface forms a planar supportfor the sample.